CN104984370A - Radiographic material and preparation method and application thereof - Google Patents
Radiographic material and preparation method and application thereof Download PDFInfo
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- CN104984370A CN104984370A CN201510465373.0A CN201510465373A CN104984370A CN 104984370 A CN104984370 A CN 104984370A CN 201510465373 A CN201510465373 A CN 201510465373A CN 104984370 A CN104984370 A CN 104984370A
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Abstract
The invention discloses an ultrasonic, magnetic resonance and fluorescent multi-mode targeted radiographic material and a preparation method and application thereof. The radiographic material is stable in physicochemical property, can produce a quicker, more accurate and high-resolution imaging effect and can be widely applied to the fields such as biological analysis, medical diagnosis and imaging.
Description
Technical field
The application relates to a kind of radiography material, is specifically related to a kind of multi-modal targeting type radiography material that can be used for ultrasonic, magnetic resonance and fluorescence.
Background technology
Cancer has become harm humans life and one of healthy major disease in world wide, and cancer has become the mankind's first cause of the death in the last thirty years, exceedes the summation of acquired immune deficiency syndrome (AIDS), tuberculosis, the lethal case of malaria.At present for treatment means i.e. discovery morning, the early treatment that cancer is best.By current medical level, 80% ~ 90% early-stage cancer patient can cure.It is reported that the cancer detection of world standard can find the infantile tumour cell of about 15mm size, and in China, can be found by instrument, the chances are the Advanced cancers cell of more than several centimetres, therefore the reduction of contrast signal of contrast agent strengthens function and seems particularly important.But the contrast agent of listing is at present only for single detecting pattern, targeting ability, is difficult to maybe to arrive tumor focus position and plays contrasting effects.
For realizing the early stage Accurate Diagnosis of tumor, reduce the toxic and side effects that repeatedly medication produces, multi-modality imaging technology is arisen at the historic moment, and it is by mutually merging to reach to learn from other's strong points to offset one's weaknesses by multiple image-forming diagnose pattern, the object of mutual supplement with each other's advantages.Along with the development of multi-modality imaging technology, for improving the clinical of novel imaging device and economic benefit, in the urgent need to the novel multi-modal contrast agent that exploitation is corresponding to multi-mode imaging system, namely only a kind of contrast agent need be used then can to realize two or more imaging functions.It can provide faster, accurate, high-res, high-resolution structure and fuction imaging.Along with the development of visual treatment technology, have imaging concurrently, a frontier research issue that targeting, the multi-modal contrast agent of even treating function have become current biological medical domain.Therefore, be the targeting ability improving diagnosing tumor efficiency, reduce toxic and side effects, promote radiography material, be necessary to develop a kind of good water solubility, the multi-modal radiography material of targeting type that radiography function is strong.
Summary of the invention
For solving the problem, an object of the application is to provide the multi-modal targeting type radiography material of a kind of ultrasonic, magnetic resonance and fluorescence, with the targeting ability improving diagnosing tumor efficiency, reduce toxic and side effects, promote radiography material.Described radiography material is the bovine serum albumin microspheres being at least enclosed with hydrophilic magnetic nanoparticle and gas A; The Schiff 's base that described bovine serum albumin microspheres surface is formed with the compound crosslink at least containing 2 aldehyde radicals containing the lysine residue in bovine serum albumin; Described bovine serum albumin microspheres surface is containing the target ligand with tumor-targeting function; Described hydrophilic magnetic nanoparticle is the coated magnetic nano-particle of hydrophilic polymer, one or more optionally in glucosan and derivant thereof, Chitosan-phospholipid complex, cellulose and its derivates of described hydrophilic polymer, one or more optionally in the magnetic metal oxide nanoparticles of tool of described magnetic nano-particle; One or more optionally in air, nitrogen, helium, argon, carbon dioxide of described gas A.
Preferably, the particle diameter of described radiography material is 0.5 μm ~ 3 μm.Further preferably, the particle size range upper limit of described radiography material is optionally from 2.5 μm, 2 μm, 1.5 μm or 1.3 μm; Optional 0.8 μm or 1 μm of particle size range lower limit.
Described hydrophilic magnetic nanoparticle is the coated magnetic nano-particle of hydrophilic polymer, and wherein the mass ratio of hydrophilic polymer and magnetic nano-particle is not less than 1: 5; Preferably, the mass ratio of hydrophilic polymer and magnetic nano-particle is 1: 4 ~ 2: 1; Further preferably, the mass ratio of hydrophilic polymer and magnetic nano-particle is 1: 3 ~ 2: 1; Further preferably, the mass ratio of hydrophilic polymer and magnetic nano-particle is 1: 2 ~ 1: 1.
Preferably, the mass percentage of the hydrophilic magnetic nanoparticle in described bovine serum albumin microspheres is 10 ~ 95%.Further preferably, the mass percentage range limit of the hydrophilic magnetic nanoparticle in described bovine serum albumin microspheres is optionally from 90%, 85%, 80%, 75%, 70%, 65%; Lower limit is optionally from 15%, 20%, 25%, 30%, 35%, 40%, 45%.
The Schiff 's base that lysine residue in bovine serum albumin contained by described bovine serum albumin microspheres surface is formed with the compound at least containing 2 aldehyde radicals, by the nitrogen-atoms in the amino containing any one aldehyde radical in the compound of 2 aldehyde radicals and lysine residue, be connected to form by-N=C-double bond.Preferably, in the Schiff 's base contained by described bovine serum albumin microspheres surface and bovine serum albumin, the molar ratio of lysine residue is not less than 1: 9; Further preferably, in the Schiff 's base contained by described bovine serum albumin microspheres surface and bovine serum albumin, the molar ratio of lysine residue is 3: 7 ~ 7: 3; Further preferably, in the Schiff 's base contained by described bovine serum albumin microspheres surface and bovine serum albumin, the molar ratio of lysine residue is 2: 3 ~ 3: 2.
Preferably, the quality accounting of the target ligand on described bovine serum albumin microspheres surface in described bovine serum albumin microspheres is 1 ~ 20%.Further preferably, the quality accounting range limit of the target ligand on described bovine serum albumin microspheres surface in described bovine serum albumin microspheres is optionally from 20%, 18%, 15%; Lower limit is optionally from 1%, 3% or 5%.
Preferably, one or more optionally in dextran, dextran derivative of described hydrophilic polymer.
In the application, described magnetic nano-particle optionally from containing ferromagnetic material, preferably, appoints one or more in the oxide nano-particles of the oxide nano-particles of the oxide nano-particles of chosen from Fe, cobalt, nickel.
In the application, described magnetic nano-particle has the particle diameter of nanoscale.According to this area public office general knowledge, nanoscale is 1nm to 100nm.Those skilled in the art, the needs that can detect according to reality, select the magnetic nano-particle of suitable yardstick in 1nm to 100nm scope.Preferably, the particle size range of described hydrophilic magnetic nanoparticle is 5 ~ 25nm.
Preferably, described magnetic nano-particle is Fe
3o
4and/or γ-Fe
2o
3nanoparticle.
Preferably, the described compound at least containing 2 aldehyde radicals optionally from carbon atom be 2 ~ 20 containing the compounds of 2 aldehyde radicals.Carbon atom is 2 ~ 20 compounds containing 2 aldehyde radicals, and refer to that carbon number is 2 ~ 20 Organic substances containing 2 aldehyde radicals, described Organic substance only can contain two aldehyde functions, also can comprise other functional groups, as fat base, carboxyl, halogenic substituent etc.Described organic structural formula can think that straight chain also can contain side chain.
Preferably, the upper limit of the described compound institute carbon atom quantity at least containing 2 aldehyde radicals is optionally from 18,15,10; The lower limit of the described compound institute carbon atom quantity at least containing 2 aldehyde radicals is optionally from 3,5,8.
Preferably, the described compound at least containing 2 aldehyde radicals is glutaraldehyde.
In the application, described in there is tumor-targeting function target ligand refer to and can combine the group of having an effect for clear and definite carcinogenic site (protein molecular of inside tumor cells, or a genetic fragment).
Preferably, the target ligand described in tumor-targeting function is the folic acid with bovine serum albumin coupling.
The another object of the application is to provide a kind of method preparing described radiography material, it is characterized in that, at least containing following steps:
A) in the system being dispersed with magnetic nano-particle, add hydrophilic polymer, after stirring, after Magneto separate, washing, obtain hydrophilic magnetic nanoparticle;
B) hydrophilic magnetic nanoparticle step a) obtained, join containing bovine serum albumin and at least contain in the liquid-phase system of compound of 2 aldehyde radicals, after mix homogeneously, with the gas of liquid-phase system surface contact in containing gas A condition under, supersound process is carried out to gas, liquid two-phase interface, obtains being enclosed with hydrophilic magnetic nanoparticle and gas A and the bovine serum albumin microspheres of surface containing Schiff 's base;
C) by the compound of the target ligand containing tumor-targeting function and step b) obtain be enclosed with hydrophilic magnetic nanoparticle and gas A and the bovine serum albumin microspheres of surface containing Schiff 's base is mixed homogeneously, obtain described radiography material through being separated.
In the application, in the preparation process of step a) hydrophilic magnetic nanoparticle, the addition of hydrophilic polymer should be able to abundant coated magnetic nanoparticle, within the scope of this, those skilled in the art according to concrete requirement, can select suitable hydrophilic polymer and the ratio of magnetic nano-particle.Preferably, the mass ratio of step a) described hydrophilic polymer and magnetic nano-particle is not less than 1: 5.Preferred scope is that the mass ratio of described hydrophilic polymer and magnetic nano-particle is 1: 2 ~ 1: 1 further.
Preferably, step a) described hydrophilic polymer be the derivant of dextran and/or dextran.
Preferably, step a) described hydrophilic magnetic nanoparticle be the magnetic nano-particle of coated dextran.
Preferably, step a) described in be dispersed with the system of magnetic nano-particle preparation at least containing following steps: in the container of gassy A, under stirring condition, ammonia is dripped to precipitating completely to the solution containing metal ion, Keep agitation a period of time, described in obtaining, be dispersed with the system of magnetic nano-particle; At least one of described metal ion optionally in divalent cobalt ion, trivalent cobalt ion, ferrous ion, ferric ion or bivalent nickel ion; Described Keep agitation a period of time is Keep agitation 0.5 ~ 24 hour at 30 ~ 80 DEG C.Preferably, Keep agitation 0.5 ~ 12 hour at 30 ~ 60 DEG C; Further preferably, Keep agitation 0.5 ~ 2 hour at 40 ~ 60 DEG C.
Preferably, step b) the described liquid-phase system containing bovine serum albumin and the compound at least containing 2 aldehyde radicals, for bovine serum albumin and the compound dissolution at least containing 2 aldehyde radicals are formed after pH is 6.5 ~ 7.5 phosphate buffered saline(PBS).
Preferably, step b) mass ratio of bovine serum albumin is 0.1 ~ 19 in described hydrophilic magnetic nanoparticle and liquid-phase system.Further preferably, in described hydrophilic magnetic nanoparticle and liquid-phase system the mass ratio range upper limit of bovine serum albumin optionally from 18,15,12 or 10; Lower limit is optionally from 0.2,0.5,1,2,5,6 or 8.
Preferably, step b) described containing bovine serum albumin with at least contain in the liquid-phase system of compound of 2 aldehyde radicals, the mass concentration of bovine serum albumin is 1 ~ 8%; The volumetric concentration of the compound at least containing 2 aldehyde radicals is 3 ~ 6%.
According to general knowledge known in this field, step b) described in supersound process is carried out to gas, liquid two-phase interface, refer in gas, liquid two-phase interface place placement frequency higher than the ultrasound wave of 20000 hertz.Preferably, the ultrasonic frequency range used is 20K ~ 25K hertz.
Preferably, step c) compound of the described target ligand containing tumor-targeting function is folic acid active ester.The active fat of described folic acid is reacted by folic acid, N-hydroxy-succinamide and dicyclohexylcarbodiimide and prepares.Folic acid active ester and bovine serum albumin, by removing the coupling reaction of amide groups, are connected to the microsphere surface of bovine serum albumin.
Preferably, step c) in, by the compound of the target ligand containing tumor-targeting function and step b) obtain be enclosed with hydrophilic magnetic nanoparticle and gas A and the bovine serum albumin microspheres of surface containing Schiff 's base is mixed homogeneously, after lucifuge reacts a period of time, obtain described radiography material through being separated.
Preferably, the time of described lucifuge reaction is 4 ~ 48 hours; Preferred scope is 8 ~ 36 hours further; Preferred scope is 12 ~ 24 hours further.
Preferably, step c) described separation method for dialysis be separated and/or column chromatography for separation.
As the application one preferred embodiment, the preparation process of described radiography material is:
(1) Fe of dextran parcel is prepared
3o
4nanoparticle: pass into nitrogen (N to 250mL conical flask
2) 10 ~ 30min is for subsequent use, takes divalent iron salt (FeCl
26H
2o) 0.25 ~ 0.35g, (divalent iron salt: trivalent iron salt=1: 1 ~ 1: 2) take appropriate trivalent iron salt, is dissolved in after mixing in 30 ~ 50mL water, is placed in N in molar ratio
2in the conical flask of protection and rapid stirring (200 ~ 1000rpm), drip 30 ~ 80mL ammonia in mixed solution with 0.2 ~ 2mL/s speed, rapid stirring 10 ~ 60min at 30 ~ 60 DEG C of temperature; Taking appropriate dextran after stirring completes in mass ratio drops in mixed solution, rapid stirring 30 ~ 120min at 30 ~ 60 DEG C of temperature; Gained mixed solution is through Magneto separate, and 8000 ~ 15000rpm centrifugation, with resuspended after washed with de-ionized water 1 ~ 3 time, drips citric acid 0.004 ~ 0.009g, 0 ~ 4 DEG C of preservation.
(2) the standby Fe being enclosed with dextran of ultrasonic cavitation legal system
3o
4magnetic nano-particle and nitrogen and the bovine serum albumin microspheres of surface containing Schiff 's base: the bovine serum albumin (BSA) taking mass fraction (1 ~ 8%) is dissolved in the PBS buffer of 1 ~ 5mL and mixes, add appropriate glutaraldehyde solution simultaneously and mix, leaving standstill 20 ~ 60sec; Drip the dextran parcel Fe of step (1) gained
3o
4mix in nanoparticle 0.5 ~ 2mL to mixed solution; Ultrasonic probe is placed in liquid phase and gas phase (N
2) top layer, interface, ultrasonic rear taking-up ultrasonic probe, collects the Fe being enclosed with dextran
3o
4magnetic nano-particle and nitrogen and the microvesicle of the bovine serum albumin microspheres of surface containing Schiff 's base 0 ~ 4 DEG C of preservation.
(3) step (2) gained bovine serum albumin microspheres surface is folacin coupled: take folic acid 0.2 ~ 0.4g, be dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 5 ~ 20mL, drip triethylamine 20 ~ 60 μ L lucifuge to stir, to drip in N-hydroxy-succinamide (NHS) 0.05 ~ 0.15g and dicyclohexylcarbodiimide (DCC) 0.1 ~ 0.2g to mixed solution after dissolving completely and continue lucifuge reaction and spend the night, filter rear gained folic acid active ester next day; Drip in the microvesicle 1 ~ 4mL collected in 0.5 ~ 2mL folic acid active ester to step (2), room temperature lucifuge reaction 30 ~ 120min, through dialysis, gained and described radiography material after post is separated.
In described step (2), the diameter of ultrasonic probe is 4 ~ 8mm.
In described step (2), ultrasonic time is 10 ~ 180sec, and effective ultrasonic time is 20 ~ 50sec.
Described step (3) folic acid: NHS: DCC mol ratio is 1: 1: 1 ~ 1: 1.5: 1.5.
According to this area public office general knowledge, described dextran to be a kind ofly made up of and the glucosan of side chain many glucose molecules, and chemical formula can be abbreviated as H (C
6h
10o
5)
xoH.
Described Schiff 's base also claims Schiff's base, containing N=C double bond.In the application, the lysine residue on bovine serum albumin microspheres surface forms Schiff 's base with the compound crosslink at least containing 2 aldehyde radicals, by the aldehyde radical carbon atom at least contained in the compound of 2 aldehyde radicals, nitrogen-atoms with the amino of the lysine residue on bovine serum albumin microspheres surface, is connected to form by-N=C-double bond.The described target ligand with tumor-targeting function, namely with the folic acid of bovine serum albumin coupling, is obtained by the amino generation coupling reaction on folic acid active ester and bovine serum albumin.
In the application, MRI is writing a Chinese character in simplified form of NMR (Nuclear Magnetic Resonance)-imaging; BSA is writing a Chinese character in simplified form of bovine serum albumin; PBS buffer is writing a Chinese character in simplified form of phosphate buffered saline(PBS); GA is writing a Chinese character in simplified form of glutaraldehyde; FA is writing a Chinese character in simplified form of folic acid.
The beneficial effect of technical scheme described in the application is:
(1) radiography material provided, has that even particle size distribution, size are controlled, the advantage such as good water solubility, good biocompatibility;
(2) radiography material provided, all can be used for magnetic resonance imaging contrast, ultra sonic imaging contrast agent, fluorescent contrast agent and the aspect such as targeted drug and cell separation;
(3) radiography material provided, has medical MRI, ultrasonic and fluoroscopic visualization function, and compared with the MRI of medically clinical practice, ultrasonic and fluorescent contrast agent, radiography performance is significantly improved, and can be used for early discovery and the diagnosis of tumor.
(4) method preparing radiography material provided, adopts gentle aqueous phase system, and method is simple, is easy to extension and produces.
Should be understood that within the scope of the technical scheme that discloses in the application, above-mentioned each technical characteristic of the application and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the application's method.The use that better implementation method described in literary composition and material only present a demonstration.
Accompanying drawing explanation
Fig. 1 is main preparation process schematic diagram in the structural representation of sample 1# and embodiment 1; A () is structural representation, (b) is preparation process schematic diagram.
Fig. 2 is the grain size distribution of sample 1#.
Fig. 3 is bovine serum albumin-glutaraldehyde fluorescence spectrum figure; (a) for excitation wavelength be the fluorescence spectrum figure of 468nm bovine serum albumin, glutaraldehyde, bovine serum albumin-glutaraldehyde; (b) for excitation wavelength be the fluorescence spectrum figure of 536nm bovine serum albumin, glutaraldehyde, bovine serum albumin-glutaraldehyde; (c) for excitation wavelength be the fluorescence spectrum figure of 630nm bovine serum albumin, glutaraldehyde, bovine serum albumin-glutaraldehyde.
Fig. 4 is the cytotoxicity analysis figure of sample 1#.
Fig. 5 is the ultra sonic imaging figure of sample 1#; A () is comparison diagram; B () is for being enclosed with the Fe of dextran
3o
4magnetic nano-particle and nitrogen and the microcapsular ultrasound image of the bovine serum albumin microspheres of surface containing Schiff 's base.
Fig. 6 is the fluorescence imaging figure of sample 1#; A () is the image under fluorescence microscope common views; B () is the green fluorescence image of fluorescence microscope under excitation wavelength is 536nm; C () is the red fluorescence image of fluorescence microscope under excitation wavelength is 630nm; D () is the merging figure of b figure and c figure.
Fig. 7 is the nuclear magnetic resonance figure of sample 1#.
Detailed description of the invention
The above-mentioned feature that the application mentions, or the feature that embodiment is mentioned can combination in any.All features that this case description discloses can with any composition forms and use, each feature disclosed in description, anyly can be provided identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.
Below in conjunction with embodiment, set forth the application further.Should be understood that these embodiments are only not used in the scope of restriction the application for illustration of the application.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
When not doing specified otherwise, raw material that the application uses, is all bought by commercial sources, directly uses without special handling.
Do not do specified otherwise, the instrument adopted in embodiment and test condition as follows:
In Sample Preparation Procedure, adopt Biosafer900-92 type Ultrasound Instrument to add ultrasound wave at gas, liquid two-phase interface place, ultrasonic wave range is 20K ~ 25K hertz.
Particle size distribution carries out material particle size test on zetasizer Nano ZS type dynamic light scattering particle size analyser.
Spectrofluorimetry carries out sample spectra analysis in Hitachi F-4600 type fluorescence spectrophotometer.
Cytotoxicity analysis utilizes mtt assay to carry out cytotoxicity analysis on UV754PC type ultraviolet-uisible spectrophotometer.
Ultra sonic imaging analysis steps on auspicious diasonograph in DP-10 type carries out sample imaging research.
Fluorescence imaging analysis carries out sample imaging research on LWD300-38LFT type inverted fluorescence microscope.
Magnetic resonance imaging analysis carries out sample imaging research on Micro MR type Low-field magnetic resonance imaging instrument.
The preparation of embodiment 1 sample 1#
(1) Fe that dextran is coated is prepared
3o
4nanoparticle: pass into nitrogen 30min to 250mL conical flask for subsequent use, take divalent iron salt (FeCl
24H
2o) 0.2982g, takes trivalent iron salt (FeCl
36H
2o) 0.5406g, is dissolved in after mixing in 30mL water, is placed in N
2in the conical flask of protection and rapid stirring (800rpm), drip 10mL ammonia in mixed solution with 1mL/s speed, rapid stirring 30min at 50 DEG C of temperature; Taking 0.15g dextran after stirring completes in mass ratio drops in mixed solution, rapid stirring 60min at 50 DEG C of temperature; Gained mixed solution is through Magneto separate, and 10000rpm centrifugation, after washed with de-ionized water 1 ~ 3 time, it is resuspended to add 30ml ultra-pure water, drips citric acid 0.006g, obtains wrapping up Fe containing dextran
3o
4the liquid of nanoparticle, in 4 DEG C of preservations.
(2) take mass fraction be 5% bovine serum albumin 0.05g be dissolved in 1mL and configure pH by disodium hydrogen phosphate dodecahydrate and two hypophosphite monohydrate sodium dihydrogens and mix in the PBS buffer of 7.2, add 120 μ L mass concentrations be 25% glutaraldehyde solution and mix simultaneously, leave standstill 60sec; What drip step (1) gained wraps up Fe containing dextran
3o
4the liquid 1mL of nanoparticle is to mixed solution and mix; Ultrasonic probe is placed in liquid phase and gas phase nitrogen interface, after ultrasonic 60sec, takes out ultrasonic probe, obtain that there is the coated Fe of parcel dextran
3o
4nanoparticle and nitrogen and the system of the bovine serum albumin microspheres of surface containing Schiff 's base.
(3) folic acid 0.25g is taken, be dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, drip triethylamine 40 μ L lucifuge to stir, to drip in N-hydroxy-succinamide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g to mixed solution after dissolving completely and continue lucifuge reaction and spend the night, filter rear gained folic acid active ester next day; Drip 0.005g folic acid active ester in step (2) gained system 2mL, room temperature lucifuge reaction 60min, after dialysis, post are separated, gained is the Fe of parcel dextran parcel
3o
4the bovine serum albumin microspheres material of Schiff 's base and folate-targeted aglucon is contained on nanoparticle and nitrogen, surface, is designated as sample 1#.The structural representation of gained sample 1# is shown in Fig. 1 (a), and Fig. 1 (b) is shown in by the schematic diagram of step (2) and (3).
The preparation of embodiment 2 sample 2#
(1) γ-Fe of dextran parcel is prepared
2o
3nanoparticle: pass into nitrogen (N to 250mL conical flask
2) 10 ~ 30min is for subsequent use, takes divalent iron salt (FeCl
26H
2o) 0.5964g, takes 1.0812g trivalent iron salt (FeCl
36H
2o), be dissolved in after mixing in 60mL water, be placed in N
2protection conical flask in and rapid stirring (1000rpm), with 1.2mL/s speed drip 60mL ammonia (20%) in mixed solution, reflux 90min at 95 DEG C of temperature; Taking 0.32g dextran after stirring completes drops in mixed solution, rapid stirring 30min at 50 DEG C of temperature; Gained mixed solution is through Magneto separate, and 12000rpm centrifugation, with adding after washed with de-ionized water 1 ~ 3 time, 60ml ultra-pure water is resuspended, drips citric acid 0.012g, obtains wrapping up γ-Fe containing dextran
2o
3the liquid of nanoparticle, in 4 DEG C of preservations.
(2) take mass fraction be 5% bovine serum albumin (BSA) 0.05g be dissolved in 1mL and configure pH by disodium hydrogen phosphate dodecahydrate and two hypophosphite monohydrate sodium dihydrogens and mix in the PBS buffer of 6.8, add 120 μ L mass concentrations be 25% glutaraldehyde solution and mix simultaneously, leave standstill 60sec; What drip step (1) gained wraps up γ-Fe containing dextran
2o
3the liquid 1mL of nanoparticle is to mixed solution and mix; Ultrasonic probe is placed in liquid phase and gas phase (N
2) top layer, interface, take out ultrasonic probe after ultrasonic 60sec, obtain that there is the coated γ-Fe of parcel dextran
2o
3nanoparticle and nitrogen and the system of the bovine serum albumin microspheres of surface containing Schiff 's base.
(3) folic acid 0.25g is taken, be dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, drip triethylamine 40 μ L lucifuge to stir, to drip in N-hydroxy-succinamide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g to mixed solution after dissolving completely and continue lucifuge reaction and spend the night, filter rear gained folic acid active ester next day; Drip 0.005g folic acid active ester in step (2) gained system 2mL, room temperature lucifuge reaction 60min, through dialysis, after post is separated, gained is the γ-Fe of parcel dextran parcel
2o
3the bovine serum albumin microspheres material of Schiff 's base and folate-targeted aglucon is contained on nanoparticle and nitrogen, surface, is designated as sample 2#.
The preparation of embodiment 3 sample 3#
(1) γ-Fe of dextran parcel is prepared
2o
3nanoparticle: pass into nitrogen (N to 250mL conical flask
2) 10 ~ 30min is for subsequent use, takes divalent iron salt (FeCl
26H
2o) 0.2982g, takes 0.5406g trivalent iron salt (FeCl
36H
2o), be dissolved in after mixing in 30mL water, be placed in N
2protection conical flask in and rapid stirring (900rpm), with 1mL/s speed drip 30mL ammonia (20%) in mixed solution, reflux 90min at 95 DEG C of temperature; Take rapid stirring 20min after 0.5g mannitol after stirring completes, take 0.32g dextran after having stirred and drop in mixed solution, rapid stirring 30min at 50 DEG C of temperature; Gained mixed solution is through Magneto separate, and 12000rpm centrifugation, after washed with de-ionized water 1 ~ 3 time, it is resuspended to add 30ml ultra-pure water, drips citric acid 0.01g, obtains wrapping up γ-Fe containing dextran
2o
3the liquid of nanoparticle, in 4 DEG C of preservations.
(2) take mass fraction be 5% bovine serum albumin (BSA) 0.05g be dissolved in 1mL and configure pH by disodium hydrogen phosphate dodecahydrate and two hypophosphite monohydrate sodium dihydrogens and mix in the PBS buffer of 7.0, add 120 μ L mass concentrations be 20% glutaraldehyde solution and mix simultaneously, leave standstill 60sec; What drip step (1) gained wraps up γ-Fe containing dextran
2o
3the liquid 1mL of nanoparticle is to mixed solution and mix; Ultrasonic probe is placed in liquid phase and gas phase (N
2) top layer, interface, take out ultrasonic probe after ultrasonic 60sec, obtain that there is the coated γ-Fe of parcel dextran
2o
3nanoparticle and nitrogen and the system of the bovine serum albumin microspheres of surface containing Schiff 's base.
(3) folic acid 0.25g is taken, be dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, drip triethylamine 40 μ L lucifuge to stir, to drip in N-hydroxy-succinamide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g to mixed solution after dissolving completely and continue lucifuge reaction and spend the night, filter rear gained folic acid active ester next day; Drip 0.004g folic acid active ester in step (2) gained system 2mL, room temperature lucifuge reaction 60min, through dialysis, after post is separated, gained is the γ-Fe of parcel dextran parcel
2o
3the bovine serum albumin microspheres material of Schiff 's base and folate-targeted aglucon is contained on nanoparticle and nitrogen, surface, is designated as sample 3#.
Embodiment 4
Proportioning raw materials, preparation process, condition in the same manner as in Example 1, only change step nitrogen into argon, and gained sample is designated as sample 4#.
Embodiment 5
Proportioning raw materials, preparation process, condition in the same manner as in Example 2, only step nitrogen is changed into carbon dioxide other, gained sample is designated as sample 5#.
Embodiment 6
Proportioning raw materials, preparation process, condition, in the same manner as in Example 1, only by the FeCl of 0.5964g
26H
2the FeCl of O and 1.0812g
36H
2o changes the CoCl of 2g into
26H
2o, changes nitrogen into helium, and gained sample is designated as sample 6#.
Embodiment 7
Proportioning raw materials, preparation process, condition, in the same manner as in Example 1, only by the FeCl of 0.5964g
26H
2the FeCl of O and 1.0812g
36H
2o changes the NiCl of 2g into
26H
2o, changes nitrogen into helium, and gained sample is designated as sample 7#.
Embodiment 8
Proportioning raw materials, preparation process, condition, in the same manner as in Example 1, only dextran is changed into the chitosan of equal quality, change nitrogen into air, gained sample is designated as sample 8#.
Embodiment 9
Proportioning raw materials, preparation process, condition, in the same manner as in Example 1, only dextran is changed into the cellulose of equal quality, glutaraldehyde is changed into the Biformyl of equivalent molar number, gained sample is designated as sample 9#.
Embodiment 10
Proportioning raw materials, preparation process, condition, in the same manner as in Example 1, only glutaraldehyde is changed into 1,8-suberic aldehyde of equivalent molar number, gained sample is designated as sample 10#.
Comparative example 1
Get mass percentage be 5% bovine serum albumin (BSA) 0.05g be dissolved in 1mL and configure pH by disodium hydrogen phosphate dodecahydrate and two hypophosphite monohydrate sodium dihydrogens and mix in the PBS buffer of 7.2, add 100 μ L glutaraldehyde solutions simultaneously and mix, leaving standstill 60sec; Ultrasonic probe (diameter 6mm, power 490W) is placed in liquid phase and gas phase (N
2) top layer, interface, take out ultrasonic probe after ultrasonic 60sec, obtain there is parcel nitrogen and the system of the bovine serum albumin microspheres of surface containing Schiff 's base.Microvesicle also 4 DEG C of preservations on collection system surface, are designated as sample 11#.
Comparative example 2
Folacin coupled BSA-GA composite: take folic acid 0.25g, be dissolved in the anhydrous dimethyl sulfoxide (DMSO) of 10mL, drip triethylamine 40 μ L lucifuge to stir, to drip in N-hydroxy-succinamide (NHS) 0.1g and dicyclohexylcarbodiimide (DCC) 0.15g to mixed solution after dissolving completely and continue lucifuge reaction and spend the night, filter rear gained folic acid active ester next day; Drip 0.005g folic acid active ester and there is parcel nitrogen and in the system 2mL of the bovine serum albumin microspheres of surface containing Schiff 's base to comparative example 1 gained, room temperature lucifuge reaction 60min, through dialysis, post obtains after being separated wrapping up nitrogen and the surperficial bovine serum albumin microspheres material containing Schiff 's base and folic acid, is designated as sample 12#.
Embodiment 11 particle size distribution
Have detected the particle size distribution of sample 1# ~ sample 10#, the particle diameter of gained sample, between 0.5 μm ~ 3 μm, is all suitable as acoustic contrast agent.With sample 1# for Typical Representative, as shown in Figure 2, within the scope of 0.8 μm ~ 1.3 μm, centralized particle diameter is at about 1.1 μm for its grain size distribution.
Embodiment 12
Respectively in comparative example 1, the bovine serum albumin in PBS buffer, glutaraldehyde and sample 11# have carried out spectrofluorimetry, and result as shown in Figure 3.In Fig. 3, (a) is 468nm bovine serum albumin, glutaraldehyde, has parcel nitrogen and the fluorescence spectrum figure of bovine serum albumin microspheres of surface containing Schiff 's base for excitation wavelength; B () is 536nm bovine serum albumin, glutaraldehyde, has parcel nitrogen and the fluorescence spectrum figure of bovine serum albumin microspheres of surface containing Schiff 's base for excitation wavelength; C () is 630nm bovine serum albumin, glutaraldehyde, has parcel nitrogen and the fluorescence spectrum figure of bovine serum albumin microspheres of surface containing Schiff 's base for excitation wavelength.Can find out, independent bovine serum albumin Pseudobulbus Bletillae (Rhizoma Bletillae) glutaraldehyde does not have fluorescent effect, after only having both to combine generation Schiff 's base, just produces fluorescence.
Embodiment 13
Respectively sample 1# ~ sample 12# is carried out to cytotoxicity detection, all illustrated low cytotoxicity.With sample 1# for Typical Representative, its cytotoxicity analysis figure as shown in Figure 4.Along with the rising of concentration, there is not obvious downward trend in cell survival rate, proves that the toxicity of 1# ~ 12# sample is low cytotoxicity.
Embodiment 14
Respectively using sample 1# ~ sample 10# as contrast agent, carry out ultrasonic contrast detection, all illustrate more significant ultrasonic contrast function.With sample 1# for Typical Representative, its ultrasonic contrast figure as shown in Figure 5.Wherein (a) for blank's image (b) for namely 1# sample wraps up the coated Fe of dextran
3o
4nanoparticle and nitrogen and the bovine serum albumin microspheres of surface containing Schiff 's base, can see in (b) blank pipe of publishing picture and be shown as white (enhancing of ultrasonic contrast function), interpret sample 1# ~ sample 10# all has obvious ultra sonic imaging function.
Embodiment 15
Respectively using sample 1# ~ sample 10# as contrast agent, carry out fluoroscopic visualization detection, all illustrate more significant fluoroscopic visualization function.With sample 1# for Typical Representative, its fluoroscopic visualization figure as shown in Figure 6.Wherein (a) is the image under fluorescence microscope common views; B () is the green fluorescence image of fluorescence microscope under excitation wavelength is 536nm; C () is the red fluorescence image of fluorescence microscope under excitation wavelength is 630nm; D () is the merging figure of b figure and c figure.Can find out under different excitation, sample can send the fluorescence such as HONGGUANG and green glow (enhancing of fluoroscopic visualization function), and interpret sample 1# ~ sample 10# all has obvious fluorescence imaging function.
Embodiment 16
Respectively using sample 1# ~ sample 10# as contrast agent, carry out magnetic resonance radiography detection, all illustrate more significant magnetic resonance radiography function.With sample 1#-3# for Typical Representative, its magnetic resonance radiography figure as shown in Figure 7.Can find out that aqueous environment is in time light grey around, sample 1#-3# all presents black (MRI T
2radiography function strengthens), interpret sample 1# ~ sample 10# all has obvious nuclear magnetic resonance function.
Embodiment 17
The relaxation rate of sample 1# ~ sample 10# is measured, all possesses more typical MRI T
2relaxation behavior.With sample 1# for Typical Representative, its relaxation rate measurement result is as shown in table 1.
Table 1
Although the application with preferred embodiment openly as above; but be not for limiting claim; any those skilled in the art are not departing from the application's spirit and scope; can make possible variation and amendment, the scope that therefore protection domain of the application should define with the application's claim is as the criterion.
Claims (10)
1. a radiography material, is characterized in that, described radiography material is the bovine serum albumin microspheres being at least enclosed with hydrophilic magnetic nanoparticle and gas A; The Schiff 's base that described bovine serum albumin microspheres surface is formed with the compound crosslink at least containing 2 aldehyde radicals containing the lysine residue in bovine serum albumin; Described bovine serum albumin microspheres surface is containing the target ligand with tumor-targeting function;
Described hydrophilic magnetic nanoparticle is the coated magnetic nano-particle of hydrophilic polymer, one or more optionally in glucosan and derivant thereof, Chitosan-phospholipid complex, cellulose and its derivates of described hydrophilic polymer, one or more optionally in the magnetic metal oxide nanoparticles of tool of described magnetic nano-particle;
One or more optionally in air, nitrogen, helium, argon, carbon dioxide of described gas A.
2. radiography material according to claim 1, is characterized in that, the particle diameter of described radiography material is 0.5 μm ~ 3 μm.
3. radiography material according to claim 1, is characterized in that, one or more optionally in dextran, dextran derivative of described hydrophilic polymer; Described magnetic nano-particle appoints one or more in the oxide nano-particles of the oxide nano-particles of the oxide nano-particles of chosen from Fe, cobalt, nickel; Described magnetic nano-particle is preferably Fe
3o
4and/or γ-Fe
2o
3nanoparticle.
4. radiography material according to claim 1, is characterized in that, the described compound at least containing 2 aldehyde radicals optionally from carbon atom be 2 ~ 20 containing the compounds of 2 aldehyde radicals; The described compound at least containing 2 aldehyde radicals is preferably glutaraldehyde; The described target ligand with tumor-targeting function is the folic acid with bovine serum albumin coupling.
5. prepare a method for radiography material described in claim 1, it is characterized in that, at least containing following steps:
A) in the system being dispersed with magnetic nano-particle, add hydrophilic polymer, after stirring, after Magneto separate, washing, obtain hydrophilic magnetic nanoparticle;
B) hydrophilic magnetic nanoparticle step a) obtained, join containing bovine serum albumin and at least contain in the liquid-phase system of compound of 2 aldehyde radicals, after mix homogeneously, with the gas of liquid-phase system surface contact in containing gas A condition under, supersound process is carried out to gas, liquid two-phase interface, obtains being enclosed with hydrophilic magnetic nanoparticle and gas A and the bovine serum albumin microspheres of surface containing Schiff 's base;
C) by containing the compound of target ligand and the step b with tumor-targeting function) obtain be enclosed with hydrophilic magnetic nanoparticle and gas A and the bovine serum albumin microspheres of surface containing Schiff 's base is mixed homogeneously, obtain described radiography material through being separated.
6. method according to claim 5, is characterized in that, the mass ratio of step a) described hydrophilic polymer and magnetic nano-particle is not less than 1: 5; Step a) described hydrophilic magnetic nanoparticle is the magnetic nano-particle of coated dextran.
7. method according to claim 5, it is characterized in that, step b) the described liquid-phase system containing bovine serum albumin and the compound at least containing 2 aldehyde radicals, for bovine serum albumin and the compound dissolution at least containing 2 aldehyde radicals are formed after phosphate buffered saline(PBS); Step c) the described compound containing the target ligand with tumor-targeting function is folic acid active ester; Step c) described separation, method is dialysis separation and/or column chromatography for separation.
8. radiography material described in any one of claim 1-4 and/or radiography material that according to any one of claim 5-7 prepared by method are in the application of fluoroscopic visualization context of detection.
9. radiography material described in any one of claim 1-4 and/or radiography material that according to any one of claim 5-7 prepared by method are in the application of ultrasonic contrast context of detection.
10. radiography material described in any one of claim 1-4 and/or radiography material that according to any one of claim 5-7 prepared by method are in the application of magnetic resonance radiography context of detection.
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| CN1088114A (en) * | 1992-09-16 | 1994-06-22 | 奈科姆成像有限公司 | The improvement of contrast agent or its correlative |
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